Our objective is to continue exploration of the hypothesis that exocytosis of lysosomal contents into biliary canaliculi is a major excretory route for hepatocyte lysosomes. We propose to define the physiological and biochemical bases for the biliary excretion of lysosomal protein. We will: a. establish the cellular origin of lysosomal protein shown by us to be present in rat bile by: (1) comparison of beta-glucuronidase purified separately from rat liver and bile; (2) comparison of the hepatic subcellular distribution and biliary excretion of three lysosomal glycosidases (beta-glucuronidase, beta-galactosidase, N-acetyl-beta-glucosaminidase) with that of 3H-Triton WR-1339, a lysosomotropic compound. b. Characterize the factors affecting the extracellular release of lysosomal protein into rat bile. We will examine the following variables: sex, age, starvation, bile flow, hormones, cyclic nucleotides, microtubule binding agents and agents known to affect microfilament function. Our hypothesis for these studies is that lysosomal movement towards and fusion with the canalicular membrane is dependent on the intracellular concentration of cyclic GMP and normal microtubule and microfilament function. c. To clarify the cellular mechanism of release of lysosomal protein into rat bile. We propose three possible mechanisms (cell injury, "membrane pinching", and membrane fusion with exocytosis), and suggest these can be distinguished by a comparison of the biliary excretion of lysomal, plasma membrane, and cytoplasmic marker enzymes. d. To determine if biliary metal (copper, iron) secretion is related to the biliary excretion of lysomal protein. We will compare the subcellular distribution and biliary excretion of lysomal protein, copper and iron in the presence and absence of hepatic copper or iron overload. e. To quantitate the biliary output of lysosomal protein in man using a duodenal perfusion technique. We will measure hourly output over 24 hours of lysosomal protein and biliary lipids.